Reconstituted Systems

The prediction of the effective concentration model has been tested in an in vitro system reconstituted with El, E2 (UbcHS), and all purified to >90%... 

Studies performed at RCRM have shown that hematopoietic and immune systems reconstitution after irradiation depends greatly on the functional abilities of the stem cells. Subset analysis and expression of CD34-i- antigens on bone marrow and peripheral blood cells were studied in Chernobyl accident clean-up workers including patients with leukemia and myelodysplasia and patients exposed to the natural levels of irradiation (table 2). 

In the catalytic cycle of CYP, reducing equivalents are transferred from NADPH to CYP by a flavoprotein enzyme known as NADPH-cytochrome P450 reductase. The evidence that this enzyme is involved in CYP monooxygenations was originally derived from the observation that cytochrome c, which can function as an artificial electron acceptor for the enzyme, is an inhibitor of such oxidations. This reductase is an essential component in CYP-catalyzed enzyme systems reconstituted from purified components. Moreover antibodies prepared from purified reductase are inhibitors of microsomal... 

Systems reconstituted from purified CYP, NADPH-cytochrome P450 reductase and phosphatidylchloline will, in the presence of NADPH and 02, oxidize xenobiotics such as benzphetamine, often at rates comparable to microsomes. Although systems reconstituted from this minimal number of components are enzymatically active, other microsomal components, such as cytochrome bs, may facilitate activity either in vivo or in vitro or may even be essential for the oxidation of certain substrates. 

Another major factor when considering whole-cell versus cell-free reactions are the overall reaction kinetics. Some enzymatic reactions utilize a complex multicomponent enzyme system. Reconstitution of the crude or purified enzyme components are not usually as effective in vitro as they are when they remain in the intracellular milieu. Whole cells have often been called little bags of enzymes. Although this is an oversimplification, it is a useful concept to consider. Whole cells sequester the enzyme components in a small but concentrated form, which is usually optimal for high efficiency. Whole cells also contain co-factors, including the systems that recycle them, and control pH and ionic strength. Altogether these factors combine to make whole cells a very useful form for the presentation and use of sensitive enzyme catalysts. 

Starting from the composition of the Sulfolobus system, reconstituted poly(U)-programmed systems have been developed from other members of the crenarchaeota (T. tenax, D. mobilis) [66] Acidianus infernus (DSM 3191), Acidianus brierleyi (formerly called Sulfolobus brierleyi), Methallosphaera sedula (Sulfolobaceae) [Sanz, J.L., unpublished), and from the euryarchaeota T. acidophilum [66,127], T celer[66] and P woesei [128]. 

Activators can only elicit a very low level of activation in a transcription system reconstituted from pure RNA polymerase 11 (pol 11) and basal factors, some types of co-factor are needed in addition. One such factor was found and termed Mediator. In an in vitro transcription system reconstituted from essentially pure factors, the mediator complex was shown to stimulate basal transcription 50-fold and to support transcriptional activation from the Gal4-VP16 and Gcn4 activating protein. The Mediator complex could also be purified in a complex with RNA polymerase II (pol II), thus forming a holo-enzyme form of pol II. 

Yamanaka T, Ota A, Okunuki K (1961) A nitrite reducing system reconstituted with purified cytochrome components of Pseudomonas aeruginosa. Biochim Biophys Acta 53 294-308... 

Leo, M.A., S. lida, and C.S. Lieber (1984). Retinoic acid metabolism by a system reconstituted with cytochrome P-450. Arch. Biochem. Biophys. 234, 305-312. 

It should be emphasized here that the four major complexes of the electron transport chain operate quite independently in the inner mitochondrial membrane. Each is a multiprotein aggregate maintained by numerous strong associations between peptides of the complex, but there is no evidence that the complexes associate with one another in the membrane. Measurements of the lateral diffusion rates of the four complexes, of coenzyme Q, and of cytochrome c in the inner mitochondrial membrane show that the rates differ considerably, indicating that these complexes do not move together in the membrane. Kinetic studies with reconstituted systems show that electron transport does not operate by means of connected sets of the four complexes. 

The initial step is the coordination of the alkyl halide 2 to the Lewis acid to give a complex 4. The polar complex 4 can react as electrophilic agent. In cases where the group R can form a stable carbenium ion, e.g. a tert-buiyX cation, this may then act as the electrophile instead. The extent of polarization or even cleavage of the R-X bond depends on the structure of R as well as the Lewis acid used. The addition of carbenium ion species to the aromatic reactant, e.g. benzene 1, leads to formation of a cr-complex, e.g. the cyclohexadienyl cation 6, from which the aromatic system is reconstituted by loss of a proton ... 

The relation between the potential-composition data for these two systems under equilibrium conditions is shown in Fig. 13. It is seen that the phase Li2hSn (Lil3Sn5) is stable over a potential range that includes the upper two-phase reconstitution reaction plateau in the lithium-silicon system. Therefore, lithium can react with Si to form the phase Li, 7 S i... 

This concept has also been demonstrated at ambient temperature in the case of the Li-Sn-Cd system [47,48]. The composition-de-pendences of the potentials in the two binary systems at ambient temperatures are shown in Fig. 15, and the calculated phase stability diagram for this ternary system is shown in Fig. 16. It was shown that the phase Li4 4Sn, which has fast chemical diffusion for lithium, is stable at the potentials of two of the Li-Cd reconstitution reaction plateaus, and therefore can be used as a matrix phase. 

Thus three lines of evidence define the rapidly dissociating receptor as the LR complex. Conditions known to uncouple R from G--first, guanine nucleotide and second, pertussis toxin—produce LR third, reconstitution of G protein restores receptor affinity, sensitivity to guanine nucleotide, and effector activation. In this sense, the ligand and binding behavior of this system is analogous to that of the beta-adrenergic receptor, where the LR and LRG complexes have already been studied with purified proteins and reconstituted membrane preparations (2,i0). 

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